We have developed yeast vacuoles as a paradigm of membrane fusion in endocytic and exocytic traffic. Each stage of this traffic, from yeast to humans, uses similar Rab, effector complex, SNAREs, and SNARE chaperones, but studies of yeast vacuole fusion have unique technical advantages: superb genetics, genomics, and biochemistry of baker's yeast, ease of visualizing the vacuole and purifying it, and the rapid and quantitative assays which we've developed for fusion and its subreactions. In addition to studying vacuole fusion in vivo and with the purified organelle in vitro, we have developed in this funding period a reconstitution of proteoliposome (RPL) fusion with all-purified components: the 4 vacuolar SNAREs, the Ypt7p Rab GTPase, its effector complex HOPS (in which the vacuolar SM protein Vps33p is one of 6 subunits), the SNARE disassembly chaperones Sec17p/Sec18p, 9 physiological vacuolar lipids, and ATP. This system has revealed basic mechanistic features of fusion which underlie each aim of our proposal: (i) The fusion pathway proceeds through defined stages; we will use our published assays of tethering and trans-SNARE complex formation, and will exploit our fluorimetric assays of content mixing, lipid mixing, permeability, and lysis, using agents that interrupt fusion to seek hemifusion (partial lipid mixing without content mixing). (ii) Diverse vacuolar lipids are needed for rapid fusion, beyond simply forming a bilayer; we will explore which are needed for peripheral membrane protein binding, tethering, trans-SNARE complex assembly, and the lipid rearrangements of hemifusion and fusion. (iii) Fusion catalysts undergo regulated cycles: 1. HOPS and Sec17/18p synergistically support fusion as SNAREs cycle between cis-complexes, the uncomplexed state, and trans-complexes, 2. Gyp7p and Ccz1/Mon1 catalyze the Rab Ypt7p cycling between GTP- and GDP-bound states, and 3. the vacuolar kinase Yck3p and unknown phosphatase(s) regulate HOPS phosphorylation. These 3 cycles are likely related, and our unique tools will allow exploration of these relationships. Understanding the vacuolar HOPS complex, discovered and studied in this work, underlies studies of human arthrogryposis, renal dysfunction, and cholestasis syndromes, caused by mutations in the human VPS33 gene. Human HOPS has recently been shown to have a unique and central role in Marburg and Ebola virus invasion of human cells. The proposed studies are thus important for both basic and clinical science.